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Leucine-specific domain modulates the aminoacylation and proofreading functional cycle of bacterial leucyl-tRNA synthetase.

Yan W, Tan M, Eriani G, Wang ED - Nucleic Acids Res. (2013)

Bottom Line: Additional analysis established that the Lys598 in the LSD is the critical residue for tRNA binding.Conversion of Lys598 to Ala simultaneously reduces the tRNA-binding strength and aminoacylation and editing capacities, indicating that these factors are subtly connected and controlled at the level of the LSD.The present work provides a novel framework of co-evolution between LeuRS and its cognate tRNA through LSD.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Molecular Biology, Center for RNA Research, Institute of Biochemistry and Cell Biology, the Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, PR China.

ABSTRACT
The leucine-specific domain (LSD) is a compact well-ordered module that participates in positioning of the conserved KMSKS catalytic loop in most leucyl-tRNA synthetases (LeuRSs). However, the LeuRS from Mycoplasma mobile (MmLeuRS) has a tetrapeptide GKDG instead of the LSD. Here, we show that the tetrapeptide GKDG can confer tRNA charging and post-transfer editing activity when transplanted into an inactive Escherichia coli LeuRS (EcLeuRS) that has had its LSD deleted. Reciprocally, the LSD, together with the CP1-editing domain of EcLeuRS, can cooperate when inserted into the scaffold of the minimal MmLeuRS, and this generates an enzyme nearly as active as EcLeuRS. Further, we show that LSD participates in tRNA(Leu) recognition and favours the binding of tRNAs harbouring a large loop in the variable arm. Additional analysis established that the Lys598 in the LSD is the critical residue for tRNA binding. Conversion of Lys598 to Ala simultaneously reduces the tRNA-binding strength and aminoacylation and editing capacities, indicating that these factors are subtly connected and controlled at the level of the LSD. The present work provides a novel framework of co-evolution between LeuRS and its cognate tRNA through LSD.

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Impact of LSD mutations on aminoacylation and editing of EcLeuRS. (A) Three-dimensional view of EcLeuRS showing the LSD motion in the aminoacylation (blue) and editing state (red) (PDB entry 4AQ7 and 4ARC). (B) Sequence alignment based on structural elements of the LeuRS LSD; the tetrapeptide linker is highlighted in green. Ec, Escherichia coli; Aa, Aquifex aeolicus; Mm, Mycoplasma mobile. (C) Aminoacylation of 10 µM EctRNALeu by 5 nM of EcLeuRS (black circle), EcLeuRS-GKDG (black square) and EcLeuRS-AAAA (black triangle). (D) Hydrolysis of 1 µM [3H]-Ile-EctRNALeu by 5 nM of EcLeuRS (black circle), EcLeuRS-GKDG (black square), EcLeuRS-AAAA (black triangle) and no enzyme (open circle) .
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gkt185-F1: Impact of LSD mutations on aminoacylation and editing of EcLeuRS. (A) Three-dimensional view of EcLeuRS showing the LSD motion in the aminoacylation (blue) and editing state (red) (PDB entry 4AQ7 and 4ARC). (B) Sequence alignment based on structural elements of the LeuRS LSD; the tetrapeptide linker is highlighted in green. Ec, Escherichia coli; Aa, Aquifex aeolicus; Mm, Mycoplasma mobile. (C) Aminoacylation of 10 µM EctRNALeu by 5 nM of EcLeuRS (black circle), EcLeuRS-GKDG (black square) and EcLeuRS-AAAA (black triangle). (D) Hydrolysis of 1 µM [3H]-Ile-EctRNALeu by 5 nM of EcLeuRS (black circle), EcLeuRS-GKDG (black square), EcLeuRS-AAAA (black triangle) and no enzyme (open circle) .

Mentions: A recent study revealed that the CP1 domain and LSD of EcLeuRS both undergo large rotations when tRNA shifts from the synthetic site to the editing active site (Figure 1A) (4). The CP1 domain rotates by 12° to open up a passage for the translocation of the 3′ end of the tRNA, while the more dynamic LSD, together with the adjacent catalytically crucial KMSKS loop, is rotated by about 33° between the aminoacylation and editing conformations. Consistently, both the CP1 domain and LSD positions move by about 19° and 35° in the TtLeuRS when comparing the aminoacylation and editing conformations (5). Another study indicated that the tRNA-triggered conformational rearrangement leads to inter-domain communication between the editing and synthetic domains of EcLeuRS (11). All these data strongly suggest that both the CP1 domain and LSD are functionally connected and cooperate during the aminoacylation and editing reactions.Figure 1.


Leucine-specific domain modulates the aminoacylation and proofreading functional cycle of bacterial leucyl-tRNA synthetase.

Yan W, Tan M, Eriani G, Wang ED - Nucleic Acids Res. (2013)

Impact of LSD mutations on aminoacylation and editing of EcLeuRS. (A) Three-dimensional view of EcLeuRS showing the LSD motion in the aminoacylation (blue) and editing state (red) (PDB entry 4AQ7 and 4ARC). (B) Sequence alignment based on structural elements of the LeuRS LSD; the tetrapeptide linker is highlighted in green. Ec, Escherichia coli; Aa, Aquifex aeolicus; Mm, Mycoplasma mobile. (C) Aminoacylation of 10 µM EctRNALeu by 5 nM of EcLeuRS (black circle), EcLeuRS-GKDG (black square) and EcLeuRS-AAAA (black triangle). (D) Hydrolysis of 1 µM [3H]-Ile-EctRNALeu by 5 nM of EcLeuRS (black circle), EcLeuRS-GKDG (black square), EcLeuRS-AAAA (black triangle) and no enzyme (open circle) .
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3643597&req=5

gkt185-F1: Impact of LSD mutations on aminoacylation and editing of EcLeuRS. (A) Three-dimensional view of EcLeuRS showing the LSD motion in the aminoacylation (blue) and editing state (red) (PDB entry 4AQ7 and 4ARC). (B) Sequence alignment based on structural elements of the LeuRS LSD; the tetrapeptide linker is highlighted in green. Ec, Escherichia coli; Aa, Aquifex aeolicus; Mm, Mycoplasma mobile. (C) Aminoacylation of 10 µM EctRNALeu by 5 nM of EcLeuRS (black circle), EcLeuRS-GKDG (black square) and EcLeuRS-AAAA (black triangle). (D) Hydrolysis of 1 µM [3H]-Ile-EctRNALeu by 5 nM of EcLeuRS (black circle), EcLeuRS-GKDG (black square), EcLeuRS-AAAA (black triangle) and no enzyme (open circle) .
Mentions: A recent study revealed that the CP1 domain and LSD of EcLeuRS both undergo large rotations when tRNA shifts from the synthetic site to the editing active site (Figure 1A) (4). The CP1 domain rotates by 12° to open up a passage for the translocation of the 3′ end of the tRNA, while the more dynamic LSD, together with the adjacent catalytically crucial KMSKS loop, is rotated by about 33° between the aminoacylation and editing conformations. Consistently, both the CP1 domain and LSD positions move by about 19° and 35° in the TtLeuRS when comparing the aminoacylation and editing conformations (5). Another study indicated that the tRNA-triggered conformational rearrangement leads to inter-domain communication between the editing and synthetic domains of EcLeuRS (11). All these data strongly suggest that both the CP1 domain and LSD are functionally connected and cooperate during the aminoacylation and editing reactions.Figure 1.

Bottom Line: Additional analysis established that the Lys598 in the LSD is the critical residue for tRNA binding.Conversion of Lys598 to Ala simultaneously reduces the tRNA-binding strength and aminoacylation and editing capacities, indicating that these factors are subtly connected and controlled at the level of the LSD.The present work provides a novel framework of co-evolution between LeuRS and its cognate tRNA through LSD.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Molecular Biology, Center for RNA Research, Institute of Biochemistry and Cell Biology, the Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, PR China.

ABSTRACT
The leucine-specific domain (LSD) is a compact well-ordered module that participates in positioning of the conserved KMSKS catalytic loop in most leucyl-tRNA synthetases (LeuRSs). However, the LeuRS from Mycoplasma mobile (MmLeuRS) has a tetrapeptide GKDG instead of the LSD. Here, we show that the tetrapeptide GKDG can confer tRNA charging and post-transfer editing activity when transplanted into an inactive Escherichia coli LeuRS (EcLeuRS) that has had its LSD deleted. Reciprocally, the LSD, together with the CP1-editing domain of EcLeuRS, can cooperate when inserted into the scaffold of the minimal MmLeuRS, and this generates an enzyme nearly as active as EcLeuRS. Further, we show that LSD participates in tRNA(Leu) recognition and favours the binding of tRNAs harbouring a large loop in the variable arm. Additional analysis established that the Lys598 in the LSD is the critical residue for tRNA binding. Conversion of Lys598 to Ala simultaneously reduces the tRNA-binding strength and aminoacylation and editing capacities, indicating that these factors are subtly connected and controlled at the level of the LSD. The present work provides a novel framework of co-evolution between LeuRS and its cognate tRNA through LSD.

Show MeSH
Related in: MedlinePlus